Biodrugs, i.e. the drugs originating from biotechnologies, play an increasingly important part in the treatment of human pathologies. These biodrugs are represented by the therapeutic proteins (enzymes, growth hormones, monoclonal antibodies, growth factors, protein vaccines), nucleic acids (siRNA, DNA, oligonucleotides), peptides (PNA) and derivatives. In certain cases, they require transporters in order to be internalized into the target cells. In recent years the internalization of the therapeutic molecules has been the subject of numerous research and development projects aimed at increasing the efficiency of the internalization of transporters, their targeting of the cells and of the organs and also reducing their potential side effects.
Thus, families of transporters have been identified, firstly based on the intracellular transfer properties of the TAT protein of the HIV virus (Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L., Pepinsky, B., and Barsoum, J. (1994) Proc Natl Acad Sci USA 91(2), 664-668; Vives, E., Brodin, P., and Lebleu, B. (1997) J Biol Chem 272(25), 16010-16017), but also penetratin originating from the third helix of the Drosophilia Antennapedia protein (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) J Biol Chem 269(14), 10444-10450), the VP22 protein of the herpes simplex virus (Elliott, G., and O'Hare, P. (1997) Cell 88(2), 223-233; Nishi, K., and Saigo, K. (2007) J Biol Chem 282(37), 27503-27517) and synthetic peptide compounds of repetitions of basic amino acids such as arginine or lysine (Matsui H, Tomizawa K, Lu Y F, Matsushita M. Curr Protein Pept Sci. (2003) April; 4(2):151-7). These natural or synthetic peptides called PTD (for Protein Transduction Domain) or CPPs (for Cell-Penetratin Peptides) have the ability to transport and transfer molecules such as peptides or nucleic acids by a cell mechanism called endocytosis. Nevertheless, the internalization by endocytosis of the therapeutic molecules can have consequences for the activity and the intracellular evolution of these molecules. In fact, it is necessary for the endocytosis vesicles to be ruptured in order to allow the therapeutic molecule to be delivered into the cell. This rupture of the membrane of the endocytosis vesicles is often carried out at an acid pH potentially leading to a modification of the structure and the activity of the therapeutic molecule associated with the transporter. On the other hand, only a small proportion of the therapeutic molecules associated with the transporters will therefore be able to escape from the endosomes in order to return to the cytoplasm reducing the effect of the molecules.
Another mechanism of internalization of the molecules into the cells consists of the formation of cellular pores. In fact, a small number of PTDs or CPPs constituted by hydrophobic amino acids (MPG, Pep-1, Pep-2, Pep-3, SSHR [Sequence Signal Hydrophobic Region derived from human FGF4 and integrin β3]) are capable of penetrating through the plasma membrane forming cellular pores. These pores, depending on their size, can thus allow the direct diffusion of the therapeutic molecule into the cytoplasm without passing through the endocytosis vesicles (Langel, Ü. (2006) Handbook of Cell-Penetrating Peptides, 2 Ed.; Hawiger J. Curr Opin Chem. Biol. 1999 February; 3(1):89-94; Yan Liu X, Robinson D, Veach R A, Liu D, Timmons S, Collins R D, Hawiger J., J Biol. Chem. 2000 Jun. 2; 275(22):16774-8). The formation of the pores, if too numerous or too large, can in certain cases prove harmful to the cell, leading to cytosol leakage to the extracellular matrix resulting in cell death.
Rothe and Lenormand (Curr.t protoc. in Protein Sci., 54: 18,11, 1-18.11.29, 2008) describe a method for producing fusion proteins comprising a segment of the ZEBRA protein (extending from the amino acid in position 170 to the amino acid in position 222) and the EGFP protein or β-galactosidase. Said fusion proteins are capable of being internalized into HeLa cells at a concentration of 0.01 μM to 0.3 μM.
The ZEBRA protein, represented by the sequence SEQ ID NO: 42, is a transcriptional activator originating from the Epstein-Barr virus. It is a protein of 245 amino acids comprising an N-terminal transactivation domain (TAD), a DNA-binding domain (DB) and a leucine zipper type dimerization domain (DIM) (FIG. 1). The C-terminal domain of said protein interacts with the leucine zipper domain leading to the formation of a hydrophobic pocket which stabilizes the ZEBRA protein/DNA complex.
Until now, the internalization routes taken by the transport peptides, known to a person skilled in the art, such as endocytosis and macropinocytosis, require significant energy expenditure in order to produce this intracellular penetration mechanism. Furthermore, this internalization by endocytosis often results in the degradation of the transported polypeptide. Only a small fraction of the transport peptides are released into the cytosol after rupture of the endosomal membrane, allowing the transported polypeptides to exert their action at cell level. As a result, on an industrial production scale, in order to ensure the efficiency of the transduction of polypeptides of interest, it is necessary to produce a large quantity of transporter and polypeptides of interest, which sometimes requires a stringent production or purification procedure, and cannot be achieved for all types of polypeptides of interest.
As a result, there is a great need to make available a transporter intended for the internalization of molecules of interest into the target cells which, on the one hand, makes it possible to transport molecules of interest into the target cells at a low concentration with high efficiency, whilst retaining the partial or total degradation of the molecules of interest inside the target cells and, on the other hand, exhibits weak cytotoxicity vis-à-vis the target cells.